We have found that 5-halomethylenetetrahydro-2-furanones and 6-halomethylenetetra-hydro-2-pyranones can act as highly effective suicide substrates for the serine proteases chymotrypsin and trypsin. These compounds act by revealing a halomethylketone group upon acyl transfer to the active site serine; during the lifetime of the acyl enzyme, this potent alkylating agent is tethered within the active site and available for reaction with nucleophilic residues. In studying these compounds, we have developed a comprehensive method for evaluating the parameters of inactivation kinetics and inactivation efficiency. In the studies we are proposing to undertake, we will use the versatile halolactonization of acetylenic acids to prepare additional inactivators of these proteases. We will use these to further our understanding of the relationships between lactone structure and the parameters of protease inactivation rate and efficiency, so as to develop inactivators that have the highest attainable efficiency and selectivity. Efforts will be concentrated on inactivators of trypsin and trypsin-like proteases. The enantiomer specificity will be determined, and radiolabeled inactivators will be used to determine inactivation stoichiometry and to identify the site of covalent attachment. Alpha-Amino-substituted lactone analogs will be prepared and converted into peptidyl derivatives to increase specificity towards particular proteases of interest. Potential suicide protease substrates embodying new reactive groups in latent form (such as acyl chlorides or nitriles, or vinylic and acetylenic ketones) will be prepared and evaluated in terms of their rate and efficiency of protease inactivation. These investigations should provide us with a potent set of protease inactivating agents, capable of selective and efficient inhibition of specific proteases. It is anticipated that these inactivators will be uniquely useful as probes for the role of proteases in normal processes of physiological regulation (e.g., fertilization, blastocyst implantation) as well as in pathological conditions (e.g., tumor invasion, metastasis, pancreatitis).